The long-range goal of this project is to understand the functional role and unusual association of phospholipids at individual binding sites on mitochondrial membrane proteins. Towards this aim, emphasis will be placed on determining the association of a unique mitochondrial phospholipid, cardiolipin, with a number of proteins known to have a functional dependence on cardiolipin for their biological function. Three types of mitochondrial membrane associated proteins will be studied: 1) the multisubunit electron transport complexes (cytochrome c oxidase and cytochrome bc1); 2) the dimeric inner membrane translocases (ADP/ATP translocase); and 3) the presequences of two cytoplasmically synthesized and mitochondrially targeted proteins (the synthetic presequences of yeast cytochrome c oxidase subunit IV and human ornithine transcarbamylase). The approach to be taken is to directly study the structural features of the cardiolipin binding site within each of these purified proteins using a series of novel synthetic cardiolipin derivatives. The synthetic cardiolipin analogues will contain chemically reactive groups, photoreactive groups (arylazides), fluorescent labels (dansyl and fluorescein) and easily detected groups (biotin). Several derivatives will be di-substituted so that cardiolipin can be used to covalently label subunits with biotin and fluorescent probes, and to crosslink subunits and sequences interacting with its apolar and polar regions. Some of these cardiolipin derivatives have been prepared during the last period of funding, others will be synthesized during the next phase of the project. Understanding the unique association of cardiolipin with one of these proteins, cytochrome c oxidase, has been one of the major objectives of our research during the past two periods of funding. The current proposal extends these studies to include other mitochondrial enzymes and proteins that are functionally dependent upon cardiolipin. By comparing the association of synthetic cardiolipin analogues with several different membrane proteins, structural features that are common among these cardiolipin dependent proteins will be determined, and the molecular basis of cardiolipin participation in mitochondrial function will be established